Unveiling a rare BRAF mutation in minimally invasive follicular thyroid carcinoma: A case report

Rationale: Molecular testing is becoming more widely used; however, the accuracy of diagnostic testing remains a primary consideration, especially for molecular testing that detects specific mutations associated with cancers. Patient concerns: A 45-year-old female without documented comorbidities presented a thyroid nodule during a routine health examination. Initial evaluation revealed a 3.8-cm nodule in the left lobe of thyroid, classified as Bethesda System category III on fine needle aspiration cytology. Genetic molecular testing detected the BRAF V600E mutation via quantitative polymerase chain reaction assay, raising concern for papillary thyroid cancer (PTC). Diagnoses: The preoperative impression was PTC based on the detection of BRAF V600E mutation. Interventions: The patient underwent thyroidectomy as well as lymph node dissection with the expectation to treat PTC. Outcomes: The final pathology unexpectedly revealed minimally invasive follicular carcinoma. Confirmatory Sanger sequencing unveiled a novel sequence variation involving nucleotide duplication within the range of 1794 to 1802, a non-V600E BRAF mutation not previously reported in follicular thyroid carcinoma. Lessons: This case study demonstrates the clinical relevance of exercising caution in molecular testing and its interpretation of results. For genetic testing used for diagnostic purposes, rigorous validation or cross-checking using different methods should always be considered to ensure appropriate interpretation of molecular results.


Introduction
Thyroid cancer stands as the most prevalent among endocrine malignancies, with an age-standardized incidence rate of 15.46 per 100,000 person-years in Taiwan for the year 2019. [1,2]hyroid cancer commonly presents oncogenic genetic alterations, each associated with specific tumor types. [3]Among these, the BRAF V600E mutation is the most frequently observed, primarily in papillary thyroid carcinoma (PTC). [4]efore to the introduction of molecular testing, indeterminate thyroid nodules frequently necessitated diagnostic surgery. [5,6]olecular genetic testing performed before thyroid nodule surgery has demonstrated effectiveness in improving diagnostic accuracy, thereby contributing to the reduction of unnecessary thyroidectomies. [6,7]However, the accuracy of diagnostic

J-FH and C-SK contributed equally to this work.
This work was supported by the National Science and Technology Council, Taiwan (MOST 110-2314-B-075-027-MY3) and the Taipei Veterans General Hospital (V110C-165, V110C-175, V111C-188, V111C-190, V112C-164).These funding agencies had no influence on the study design, data collection or analysis, decision to publish or preparation of the manuscript.
The patient described in this case report has provided written informed consent for the publication of their case details, including any accompanying images.Every effort has been made to ensure patient confidentiality, and identifying information has been omitted or anonymized wherever possible.
The authors declare that there are no conflicts of interest associated with the research.
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
The project refrained from engaging in any illegal commercial transactions, utilizing organs or materials sourced from executed prisoners, or participating in other unethical practices.
testing remains a primary consideration, especially for molecular testing that detects specific mutations associated with cancers.Herein, we introduce a case involving the Bethesda system category III thyroid nodule harboring a positive BRAF V600E mutation detected via quantitative polymerase chain reaction (qPCR) assay, followed by thyroidectomy and lymph node dissection. [8]Nevertheless, the definitive pathology report yielded an unexpected diagnosis of indolent follicular thyroid carcinoma (FTC) accompanied by a rare non-V600E BRAF mutation, confirmed by Sanger sequencing.Moreover, we made a brief review of rare BRAF mutations in thyroid carcinomas.

Case presentation
The Institutional Review Board of Taipei Veterans General Hospital approved the study (IRB No: 2023-01-020CC), and the research was performed with written informed consent and in accordance with the Declaration of Helsinki.
A 45-year-old Taiwanese female came to the outpatient clinic because she had a left thyroid nodule for 2 years.She had no major underlying medical conditions.Taking her family history, her mother and younger sister were diagnosed with hypothyroidism.
The blood panel affirmed her euthyroid status without thyroid autoantibodies in terms of antithyroglobulin antibodies, antithyroid peroxidase antibody, and thyroid-stimulating immunoglobulin but elevated thyroglobulin level (Table 1).Sonographic examination of the thyroid revealed a 3.8-cm mass within the left thyroid lobe, featuring solid composition, hypoechogenicity, wider-than-taller shape, smooth margin, and absence of echogenic foci categorized as Thyroid Imaging Reporting and Data System [9] 4 with no significant interval change in the recent 2 years (Fig. 1).Fine needle aspiration (FNA) was performed, with cytological analysis with enlarged nucleus and some microfollicular arrangement yielding a verdict of TBS category III: atypia of undetermined significance. [8]urther molecular genetic testing of FNA samples stored in CytoRich Red Collection Fluid (Thermo Fisher Scientific, Waltham, MA, USA) was conducted.The isolated DNA was quantified using Qubit™ dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), and mutationspecific qPCR experiments were performed with ThyroSCAN Cancer Diagnostics Kit (Quak BioTechnology, Taiwan) for the detection of five single nucleotide variants (BRAF V600E, NRAS Q61R, NRAS Q61K, HRAS Q61R, HRAS Q61K).The BRAF V600E mutation was detected with a cycle threshold (Ct) value of 24.4,falling below the predetermined cutoff Ct value of 25 based on the standard curve method, resulting in the positive identification of BRAF V600E and raising concern for PTC.A preoperative neck computed tomography scan showed a 3.8-cm mass situated within the left thyroid lobe, while no regional lymphadenopathy was identified (Fig. 2).Therefore, the decision was made to proceed with a left lobectomy along with central lymph node dissection, and the patient underwent the procedure smoothly without any adverse events.The histological examination revealed a 3.7 × 3.5 × 1.5 cm tumor composed of solid pattern of microfollicles with complete encapsulation exhibiting minimal capsular invasion in one focus while showing no evidence of vascular or lymphatic invasion, nor perineural invasion, and lacking the nuclear characteristics typically associated with PTC, such as multifocal nuclear enlargement, nuclear overlapping, chromatin clearing, and nuclear grooves.Additionally, immunohistochemical analysis demonstrated negative staining for BRAF (clone: VE1, Ventana, Oro Valley, AZ, USA) and NRAS Q61R (clone: RBT-NRAS, Bio SB, Goleta, CA, USA) (Fig. 3).The final pathology report indicated minimally invasive FTC, staged as pT2N0M0.
For the discrepancy of preoperative qPCR and immunohistochemical staining, formalin-fixed paraffin-embedded tissue samples were subjected to perform Sanger sequencing of BRAF exon 15, uncovering a nucleotide duplication within the coding sequence spanning from 1794 to 1802, resulting in the protein alteration identified as V600_K601insNTV (Fig. 4).Although the final pathology results differed from the initial impression, the patient still appreciated that the malignancy was treated based on preoperative molecular genetic testing.

Discussion
We present a case featuring an indeterminate thyroid nodule whose FNA molecular testing showed BRAF V600E positivity, whereas, the final pathology surprisingly revealed a follicular carcinoma.Further molecular analysis of pathologic samples unveiled a rare BRAF V600_K601insNTV mutation, previously reported in only one case of follicular variant of PTC. [10]o our best knowledge, this is the first reported case of FTC with BRAF V600_K601insNTV mutation.The prevalence of thyroid cancers with rare BRAF mutations is summarized in Table 2.
[12][13] Additionally, another infrequent BRAF mutation, BRAF G469A, is barely observed in thyroid tumors but primarily occurs in malignant struma ovarii with thyroid-type carcinoma morphology. [35]Based on current literature, thyroid carcinomas harboring rare BRAF mutations are generally linked to less aggressive tumor behavior, characterized by unifocality, complete encapsulation, intrathyroidal presentation with a low incidence of lymph node metastases, and less advanced   tumor staging at diagnosis, consistent with the findings in our patient. [10]he pathogenesis of PTC is influenced by the mitogenactivated protein kinase (MAPK) pathway, which plays a crucial role in regulating cellular proliferation, differentiation, and programmed cell death. [36]Within this pathway, the predominant driver mutation observed in PTC occurs in the 15th exon of the BRAF gene, resulting in a substitution of valine with glutamate at codon 600, known as BRAF V600E. [26]Moreover, RAS mutations, mutually exclusive with BRAF V600E, also trigger the activation of the MAPK pathway. [37]Despite both involvement in the MAPK pathway, BRAF and RAS mutations lead to different degrees of activation within the signaling cascade. [36,38]pERK1/2 serves as an indicator of MAPK pathway activity, with higher expression levels observed in BRAFmutated tumors compared to RAS-mutated tumors. [36,38]This disparity may elucidate the association of BRAF V600E with a more unfavorable prognosis, including heightened disease recurrence, increased tumor aggressiveness, extrathyroidal spread, and the development of local and distant lymph node metastases. [39]Conversely, RAS mutations are often associated with less aggressive behavior, frequently presenting as tumors with a follicular growth pattern, encapsulation, minimal invasiveness, and a reduced likelihood of recurrence. [38,40]ven that all BRAF mutations other than BRAF V600E exhibit RAS-like behavior, it may explain why thyroid cancers with rare BRAF mutations exhibit an indolent nature and a higher frequency of the follicular variant. [36]While our case report unveiled a novel BRAF mutation in FTC displaying RAS-like characteristics, additional research is required to clarify the involvement of other pathways implicated in the pathogenesis of PTC with rare BRAF mutations, that is, the phosphatidylinositol-3 kinase/serine-threonine protein kinase AKT signaling pathway and PAX8/peroxisome proliferatoractivated receptor γ rearrangement.Furthermore, the effects of V600_K601insNTV on the MAPK signaling pathway and its responsiveness to BRAF inhibitors remain unexplored and warrant further investigation.
Molecular markers have emerged as a promising approach in cases of indeterminate thyroid FNA specimens, offering crucial insights to guide decisions regarding primary surgical treatment. [5,41]The prevalence of BRAF V600E mutation differs in different subtype of thyroid cancer, depending on the detection method and size of study cohort.The BRAF V600E mutation is detected in approximately 30-70% of PTC cases and 30% to 40% of anaplastic thyroid carcinoma cases. [39,40,42]Recent reports showed BRAF V600E mutation was detected in around 10% of FTC according to the 2017 WHO classification, but not detected in FTC according to the 2023 WHO classification, in which the oncocytic carcinomas of the thyroid are distinguished from FTC. [43,44] Given its marked specificity, thyroidectomy is recommended when BRAF V600E mutation is present. [45,46]he recognition of BRAF mutation as a potential predictive indicator for occult metastasis also prompts consideration of elective neck dissection in region VI. [45,46]Nonetheless, implementing neck dissection based on preoperative identification of the BRAF V600E mutation in cN0 PTC still remains controversial, primarily due to concerns regarding potential surgical complications and the limited positive predictive value of disease recurrence. [47]In retrospect, the necessity of the central lymph node dissection procedure for our patient may warrant reconsideration.However, it is essential to highlight that despite the potential for false positives, preoperative FNA cytology molecular genetic analysis significantly contributes to the early intervention of malignant thyroid nodules.CV-PTC = classic variant of papillary thyroid carcinoma, FA = follicular adenoma, FTC = follicular thyroid carcinoma, FV-PTC = follicular variant of papillary thyroid carcinoma, HNV-PTC = hobnail variant of papillary thyroid cancer, PDTC = poorly differentiated thyroid carcinoma, PTC = papillary thyroid carcinoma, TSV-PTC = trabecular-solid pattern of growth papillary thyroid carcinoma.
In this study, the Ct value of BRAF V600E detected by mutation-specific qPCR was 24.4, which is close to the cutoff Ct value of 25.Although the false positive rate of qPCR detection methods ranges from 0.08% to 5.4%, [48] our results certainly need further validation.BRAF V600E mutation detection methods have been greatly developed, including digital PCR.Locked nucleic acid probe-based digital PCR can be used to further validate our results, as it has been shown to accurately detect BRAF V600E variants with higher sensitivity than immunohistochemistry and Sanger sequencing. [49]In addition, qPCR and digital PCR methods are mainly used to detect known biomarkers and are limited in discovering new mutations.

Conclusions
The case presented highlights the critical need to thoroughly evaluate molecular genetic testing results in thyroid nodule FNA cytology.While the BRAF V600E mutation is commonly linked to PTC, its presence does not exclude other types of thyroid malignancies.Additionally, the identification of a rare BRAF mutation in our case, previously unreported in FTC, exhibited an indolent behavior consistent with its RAS-like characteristics.

Figure 1 .
Figure 1.The ultrasonographic examination showed a 3.8-cm left thyroid mass, featuring solid composition, hypoechogenicity, wider-than-taller shape, smooth margin, and absence of echogenic foci categorized as thyroid imaging reporting and data system (TI-RADS) 4 (A) in Aug 2021 (B) in Jun 2023, with no discernible interval change.

Figure 2 .
Figure 2. Computed tomography (CT) scan revealed a 3.8-cm mass situated within the left thyroid lobe without apparent signs of regional lymphadenopathy.

Figure 4 .
Figure 4. Electropherogram of the duplicated sequence following GTGAA.One represents the mutated allele with a nucleotide change: c.1794_1802dup (p.V600_K601insNTV), while the other corresponds to the wild-type allele.

Table 1
Thyroid function, tumor markers, and autoantibody data.

Table 2
Summary of thyroid with rare BRAF mutation.